Andrew D. Price

A Microreactor with Thousands of Subcompartments: Enzyme‐Loaded Liposomes within Polymer Capsules

Fully loaded: Noncovalent anchoring of liposomes into polymer multilayered films with cholesterol-modified polymers allows the preparation of capsosomes-liposome-compartmentalized polymer capsules (see picture). A quantitative enzymatic reaction confirmed the presence of active cargo within the capsosomes and was used to determine the number of subcompartments within this novel biomedical carrier system.

Nanoporous colloids: building blocks for a new generation of structured materials

Porous colloidal particles have recently attracted a renewed interest in both fundamental and applied research. The large internal surface area and adsorbent potential of porous particles have led to their widespread application in diverse fields, including sensing, catalysis, drug delivery, and separations. This review highlights recent advancements in the adsorption of molecular species and nanoparticles into nanoporous colloids, and the subsequent formation of colloidal composites, particle replicas, and ordered particle arrays. We begin with a brief introduction to the synthesis of porous colloidal particles that are commonly employed, followed by a survey of the use of porous particles as adsorbents for the immobilization and entrapment of nanoscale guest materials. Next, recently developed methods to control the release of entrapped materials as well as their templated use for assembling hollow capsules with entrapped guest materials are detailed. We subsequently highlight recent developments in the templated synthesis of nanoporous particles with an emphasis on mesoporous silica replicas. Finally, new opportunities for using porous colloidal particles in the assembly of ordered materials are discussed.

Triggered Enzymatic Degradation of DNA within Selectively Permeable Polymer Capsule Microreactors

The compartmentalization of reactions, a strategy developed by living cells, enables multiple, simultaneous reactions to be carried out within spatially separated reaction vessels. Various strategies for the compartmentalization of biochemical reactions in vitro have been applied, including their confinement in liposomes, polymersomes, water-in-oil emulsions, and polyelectrolyte capsules. A greater challenge for synthetic microreactors is the possibility of continuous reactions, for which the design of the vessel must accommodate for the constant intake of reagents and release of products. In the cell, these processes are often mediated by transmembrane proteins; however, examples of synthetic microreactors with continuous-reaction capabilities are scant. The semipermeable nature of polymer capsules formed by layer-by-layer (LbL) assembly enables the selective confinement or exclusion of molecules, primarily on the basis of their size: Small molecules, including ions and monomers, diffuse freely across the capsule walls, whereas larger macromolecules and crystals are diffusion-limited. This feature makes LbL capsules attractive candidates for use as synthetic microreactors. However, the field of encapsulated catalysis with these capsules is still in its infancy. The few successful studies reported have focused on the catalytic conversion of small molecules (e.g. carbonates, peroxides, phenols). Herein, we report an essential step towards the mimicry of cellular processes with synthetic LbL capsules: a triggered encapsulated enzymatic reaction with a nucleic acid substrate. Within the living cell, the lysosomal degradation of proteins and nucleic acids is among the most prominent examples of compartmentalized enzymatic reactions. We have carried out this reaction in a synthetic microreactor by coencapsulating double-stranded DNA (dsDNA) and the enzyme DNase I in multilayered polymer capsules. We demonstrate herein that the semipermeable nature of the microreactor affords control over the reaction with external chemical stimuli, and that by conducting the enzymatic reaction within the polymer capsules with fluorescently labeled substrate DNA, the reaction can be monitored directly within each individual capsule by the high-throughput technique flow cytometry (FC). This reaction is the first example of a triggered, continuous chemical reaction for the modification of DNA within a polymer reaction vessel. Bimodal mesoporous silica (BMS) particles were used for biomolecule immobilization and as templates for LbL assembly. The surface modification of BMS particles with a primary amine imparted a positive charge to the particles and thus created an electrostatic attraction between the particles and the introduced fluorescently labeled dsDNA. Confocal laser scanning microscopy (CLSM) images of the particles indicated the infiltration of DNA into the particles (see the Supporting Information). Subsaturation coverage of the particle surfaces by DNA, as confirmed by microelectrophoresis, was essential for the subsequent assembly of the polymer multilayers. The infiltration of DNase I, a globular protein of approximate dimensions 4.5 4.0 3.5 nm, proceeded for 1 h following charge reversal by poly(methacrylic acid) (PMA; Figure 1). The amount of adsorbed enzyme was determined to be (0.23 0.03) U of DNase I per milligram of silica by using a microbicinchoninic acid protein assay. Subsequent polymer multilayers were assembled onto the particles through hydrogen bonding between a thiol-modified PMA (PMASH) and poly(vinyl pyrrolidone) (PVPON) by alternating the adsorption of the polymers at pH 4.0. PMASH cross-linking and removal of the silica cores yielded intact polymer capsules, as confirmed by transmission electron microscopy (TEM) and differential interference contrast (DIC) microscopy (see the Supporting Information). Elevation of the pH value to 7 caused the removal of PVPON from the capsule walls by disrupting its hydrogen bonding to PMASH to produce single-component PMA capsules [14] with the nucleic acid chains confined within the interior of the capsules (Figure 2a). Although some enzyme leakage may occur from the capsules following core dissolution, the affinity of DNase I for dsDNA is likely to prevent any significant amount of the enzyme from escaping from the capsules. DNase I is an endonuclease with a strong affinity for dsDNA. The catalytic nuclease activity of DNase I requires the presence of divalent cations. Specifically, magnesium and calcium ions were shown to have a synergistic effect: A highly active enzyme–divalent cation complex was formed at pH 7.5. In the absence of these ions, the enzyme remained inactive. Their introduction is an effective trigger for enzymatic activity. In the absence of Mg and Ca, CLSM images showed that the interior of the capsules was filled with DNA (Figure 2a). The capsules showed negligible leakage of DNA for at least 72 h. However, the addition of divalent cations and the elevation of the temperature to 35 8C led to activation of [*] Dr. A. D. Price, Dr. A. N. Zelikin, Dr. Y. Wang, Prof. F. Caruso Centre for Nanoscience and Nanotechnology Department of Chemical and Biomolecular Engineering The University of Melbourne, Parkville, Victoria 3010 (Australia) Fax: (+ 61)3-8344-4153 E-mail: fcaruso@unimelb.edu.au

Triggered cargo release by encapsulated enzymatic catalysis in capsosomes.

We report the coencapsulation of glutathione reductase and disulfide-linked polymer-oligopeptide conjugates into capsosomes, polymer carrier capsules containing liposomal subcompartments. The architecture of the capsosomes enables a temperature-triggered conversion of oxidized glutathione to its reduced sulfhydryl form by the encapsulated glutathione reductase. The reduced glutathione subsequently induces the release of the encapsulated oligopeptides from the capsosomes by reducing the disulfide linkages of the conjugates. This study highlights the potential of capsosomes to continuously generate a potent antioxidant while simultaneously releasing small molecule therapeutics.

A Biomolecular “Ship-in-a-Bottle”: Continuous RNA Synthesis Within Hollow Polymer Hydrogel Assemblies

2010 WILEY-VCH Verlag Gm Synthetic counterparts to cellular compartments remain far from the complexity of living systems but hold tremendous promise for advancing studies into the synthesis, confinement, and delivery of biomolecules. Of all biomolecular candidates, RNA could benefit from encapsulation as it spans a range of cellular functions including information storage, regulation, and catalysis. RNA has proven to be a potent therapeutic for the modification of cellular function, yet unprotected, it remains highly susceptible to degradation. A practical and biomimetic approach to RNA encapsulation is the enzyme-catalyzed synthesis of RNA within the confines of a drug delivery vehicle, which has the potential to minimize the handling of RNA, bypassing isolation and purification steps. To date, the most successful examples of encapsulated transcription exploit liposomes and emulsions, yet controlled RNA loading and their subsequent use for the cellular internalization of the newly synthesized RNA have not been accomplished. Herein, we describe the use of micrometer-sized, monodisperse polymer hydrogel capsules (HCs) for the first successful example of encapsulated de novo RNA synthesis and subsequent cellular internalization of the RNA. The capsules act as both microreactors and drug carriers. Unlike other methods of encapsulated RNA transcription, polymer HCs also allow real-time monitoring of RNA synthesis and, therefore, precise control over the encapsulated RNA concentration via the reaction time. The recent development of polymer capsules produced via the layer-by-layer (LbL) deposition of polymers has provided a novel platform for encapsulated catalysis with a number of advantages, including precise control over the capsule size and permeability based on both the size and/or the charge of the solutes. Successful examples of DNA synthesis, hybridization, and degradation within the confines of polymer capsules highlight the potential of these assemblies as host compartments for biomolecular transformations. In addition, their shape, stability, and size endow them with characteristics suitable for the delivery of molecular therapeutics. The successful synthesis of RNA within the confines of a polymer HC represents a facile technique for the controlled capsule loading of RNA, en route to a drug-delivery platform for RNA therapeutics. The encapsulation of two macromolecules is essential for the synthesis of RNA within the interior of a polymer capsule: an RNA polymerase and a double-stranded DNA (dsDNA) template containing a specific promoter sequence required for enzyme binding and the initiation of RNA transcription. We have recently encapsulated DNA into poly(methacrylic acid) (PMA) HCs through LbL assembly. The PMA HCs entrap DNA through a combination of size exclusion and electrostatic repulsion. Herein, we exploit the encapsulated dsDNA to template the transcription of RNA by T7 RNA polymerase (T7Pol) (Scheme 1). T7Pol has sufficient diffusivity through the walls of PMA HCs, possibly via the ‘‘relay race’’ mechanism described for protein– hydrogel interactions. Once inside, the T7Pol binds the promoter sequence in the DNA template and is immobilized inside the capsule. Due to their small size, individual ribonucleotides freely diffuse into the PMA HCs, where T7Pol assemble them into single-stranded RNApolymers. Similar to the DNA template, the size, shape, and charge of the newly synthesized RNA polymers ensure they remained trapped within the capsules. The strategy outlined above (Scheme 1) was used to assemble PMA HCs (diameter of 4.35 0.25mm), each containing 9000 copies of a 777-base pair (bp) dsDNA polymerase chain reaction (PCR) product. The PCR products were designed to either include (þT7DNA) or exclude (–T7DNA) the T7 promoter sequence at the 50-end of the sense strand. Approximately 10 capsules were added to a 50mL transcription reaction supplemented with 0.1 nmol of fluorescently labeled uridine50-triphosphate (UTP, green) and the mixture was incubated at 37 8C. A concentration of 50 units of T7Pol in the 50mL reaction was sufficient to enable diffusion of enzyme into the core of the PMA HCs and initiate transcription. Confocal laser scanning microscopy (CLSM) was used to image fluorescently labeled HCs (red) following 3 h of incubation. Newly synthesized RNA was clearly visible in the interior of the PMA HCs containing DNA with the T7 promoter sequence (Fig. 1a), however, no fluorescencewas observed in the PMAHCs containing DNA without the T7 promoter sequence (Fig. 1b). An identical reaction was performed using 1.35 0.15mm PMA HCs, each containing 500 copies ofþT7DNA. Flow cytometry was used to measure the progress of the reaction within the PMA HCs (Fig. 1c), allowing real-time monitoring of RNA synthesis. Synthesis was slow in the first hour but then increased to a linear rate of synthesis between 1.5 h and 5 h, and finally leveled off over the next 24 h. The initial slow rate of synthesis possibly reflects the time required for the T7Pol to diffuse into the capsules and bind to the T7 promoter sequences.

Subcompartmentalized Polymer Hydrogel Capsules with Selectively Degradable Carriers and Subunits

Subcompartmentalized hydrogel capsules (SHCs) with selectively degradable carriers and subunits are designed for potential applications in drug delivery and microencapsulated biocatalysis. Thiolated poly(methacrylic acid) and poly(N-vinyl pyrrolidone) are used to assemble 3-microm-diameter carrier capsules and 300-nm-diameter subunits, independently stabilized by a diverse range of covalent linkages. This paper presents examples of SHCs with tens of subcompartments and their successful drug loading, as well as selective degradation of the SHC carrier and/or subunits in response to multiple chemical stimuli.

Poly(Methacrylic Acid) Polymer Hydrogel Capsules: Drug Carriers, Sub-compartmentalized Microreactors, Artificial Organelles

Multilayered polymer capsules attract significant research attention and are proposed as candidate materials for diverse biomedical applications, from targeted drug delivery to microencapsulated catalysis and sensors. Despite tremendous efforts, the studies which extend beyond proof of concept and report on the use of polymer capsules in drug delivery are few, as are the developments in encapsulated catalysis with the use of these carriers. In this Concept article, the recent successes of poly(methacrylic acid) hydrogel capsules as carrier vessels for delivery of therapeutic cargo, creation of microreactors, and assembly of sub-compartmentalized cell mimics are discussed. The developed technologies are outlined, successful applications of these capsules are highlighted, capsules properties which contribute to their performance in diverse applications are discussed, and further directions and plausible developments in the field are suggested.

Poly(N-isopropylacrylamide) Surfactant-Functionalized Responsive Silver Nanoparticles and Superlattices

Metal nanoparticles exhibit unique optical characteristics in visible spectra produced by local surface plasmon resonance (SPR) for a wide range of optical and electronic applications. We report the synthesis of poly(N-isopropylacrylamide) surfactant (PNIPAM-C18)-functionalized metal nanoparticles and ordered superlattice arrays through an interfacial self-assembly process. The method is simple and reliable without using complex chemistry. The PNIPAM-C18-functionalized metal nanoparticles and ordered superlattices exhibit responsive behavior modulated by external temperature and relative humidity (RH). In situ grazing-incidence small-angle X-ray scattering studies confirmed that the superlattice structure of PNIPAM-C18 surfactant-functionalized nanoparticle arrays shrink and spring back reversibly based on external thermal and RH conditions, which allow flexible manipulation of interparticle spacing for tunable SPR. PNIPAM-C18 surfactants play a key role in accomplishing this responsive property. The ease of fabrication of the responsive nanostructure facilitates investigation of nanoparticle coupling that depends on interparticle separation for potential applications in chemical and biological sensors as well as energy storage devices.

Magnetic relaxometry as applied to sensitive cancer detection and localization

Abstract Background: Here we describe superparamagnetic relaxometry (SPMR), a technology that utilizes highly sensitive magnetic sensors and superparamagnetic nanoparticles for cancer detection. Using SPMR, we sensitively and specifically detect nanoparticles conjugated to biomarkers for various types of cancer. SPMR offers high contrast in vivo, as there is no superparamagnetic background, and bones and tissue are transparent to the magnetic fields. Methods: In SPMR measurements, a brief magnetizing pulse is used to align superparamagnetic nanoparticles of a discrete size. Following the pulse, an array of superconducting quantum interference detectors (SQUID) sensors detect the decaying magnetization field. NP size is chosen so that, when bound, the induced field decays in seconds. They are functionalized with specific biomarkers and incubated with cancer cells in vitro to determine specificity and cell binding. For in vivo experiments, functionalized NPs are injected into mice with xenograft tumors, and field maps are generated to localize tumor sites. Results: Superparamagnetic NPs developed here have small size dispersion. Cell incubation studies measure specificity for different cell lines and antibodies with very high contrast. In vivo animal measurements verify SPMR localization of tumors. Our results indicate that SPMR possesses sensitivity more than 2 orders of magnitude better than previously reported.

Controlled polymer monolayer synthesis by radical transfer to surface immobilized transfer agents

Radical chain transfer from solution-generated free radicals to thiol transfer agents bound to the surfaces of planar silicon and silica particles was investigated as a method to generate surface-bound polymer brushes. Both conventional radical and reversible addition–fragmentation chain transfer (RAFT) polymerizations created polymer films with molecular weights dependent on the details of the reaction. In the presence of high free radical concentrations or RAFT agent modifiers, the surface had minimal influence on the growth of grafted polymer chains following the initial radical transfer to the surface. Notably, excellent control of film thickness was achieved in the absence of surface-bound initiators or RAFT agents, thereby simplifying the synthesis of immobilized brush architectures. On the other hand, low concentrations of solution free radicals in conventional radical polymerization generated grafted polymers with lengths varying considerably from polymers in solution. Despite the simplicity and versatility of this technique for creating polymer-modified surfaces by surface initiated polymerization (SIP), it has been under-utilized; likely due to the absence of a framework for the rational design of the brush. The details of this investigation should allow surface modification by polymer films to become accessible to researchers without the need to create complex precursor surface chemistries.